Non toxic compositions and methods useful for the extraction of nucleic acids

ABSTRACT

This invention relates to safe and effective methods for the extraction of nucleic acids. In particular, methods are described for isolating nucleic acid from a sample containing a biological mixture of nucleic acids and other biological compounds wherein the sample is combined with an extraction solution containing at least one organic compound such as benzyl alcohol or a benzyl alcohol derivative to form an aqueous and non-aqueous phase. The nucleic acid is isolated from the aqueous phase. Preferably, the resulting combined solution also contains bentonite, as defined below. Typically, the sample will first be combined with a lysing agent before extraction. The lysing agents preferred are chaotropic salts such as guanidinium hydrochloride and guanidinium isothiocyanate.

This is a continuation of application Ser. No. 07/900,379, filed Jun.17, 1992, now abandoned, which is a continuation of application Ser. No.07/649,389, filed Feb. 1, 1991, now U.S. Pat. No. 5,130,423, which is acontinuation-in-part of Ser. No. 07/552,745, filed Jul. 13, 1990, nowabandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to compositions and assay methods for theextraction and hybridization of nucleic acids. In particular, thisinvention relates to compositions and methods to extract nucleic acidsfrom cells in complex biological samples or specimens without the use oftoxic compounds, such as phenol and/or chloroform, primarily through theuse of benzyl alcohol or benzyl alcohol derivatives. The novelcompositions and methods described here are very effective in theextraction and purification of nucleic acids.

2. Brief Description of the Relevant Art

Organic solvents such as phenol and chloroform are traditionally used intechniques employed to isolate nucleic acid from procaryotic andeucaryotic cells or from complex biological samples. Nucleic acidisolations typically begin with an enzymatic digest performed withproteases followed by cell lysis using ionic detergents, and thenextraction with phenol or a phenol/chloroform combination. The organicand aqueous phases are separated and nucleic acid which has partitionedinto the aqueous phase is recovered by precipitation with alcohol.However, phenol or a phenol/chloroform mixture is corrosive to humanskin and is considered as hazardous waste, which must be carefullyhandled and properly discarded. Further, the extraction method is timeconsuming and laborious. Marmur, J. Mol. Biol. 3:208-218 (1961),describes the standard preparative procedure for extraction andpurification of intact high molecular weight DNA from procaryoticorganisms using enzymatic treatment, addition of a detergent, and theuse of an organic solvent such as phenol or phenol/chloroform. Chirgwinet al., Biochemistry 18:5294-5299 (1979) described the isolation ofintact RNA from tissues enriched in ribonuclease by homogenization inguanidinium thiocyanate and 2-mercaptoethanol, followed by ethanolprecipitation or by sedimentation through cesium chloride.

Further, the use of chaotropic agents such as guanidinium thiocyanate(GnSCN) are widely used to lyse and release nucleic acid from cells intosolution, largely due to the fact that chaotropic salts inhibitnucleases and proteases. However, it has proved difficult to isolate thenucleic acids from these chaotropic salt solutions due to theincompatibility of the chaotropes with ionic detergents and theinability to easily partition the nucleic acid into an aqueous phase,given the use of such high molar concentrations of salt.

The ability to effectively inhibit nucleases during nucleic acidisolation procedures is paramount, especially when the starting materialis complex, such as feces or blood. In 1959, Brownhill et al. reportedthat bentonite was an inhibitor of nucleases (Brownhill et al., Biochem.J. 73:434 (1959)). Fraenkel-Conrat et al. later developed a procedurefor the use of bentonite to inhibit ribonuclease in a procedure topurify tobacco mosaic virus (Fraenkel-Conrat et al., Virology 14:54-58(1961)). Subsequent researchers reported the use of bentonite incombination with phenol and chloroform in the reduction of ribonucleaseactivity during the isolation of RNA (Jacoli et al., Can. J. Biochem.51:1558-1565 (1973); Griffin et al., Anal. Biochem. 87:506-520 (1978);Grady et al., Anal. Biochem. 101:118-122 (1980)). It has also beenreported that DNase 1 and α-amylase can be made RNAse-free by treatmentwith bentonite (Garrett et al., Anal. Biochem. 52:342-348 (1973)).

All of the above traditional nucleic acid extraction procedures, thus,require the use of toxic compounds such as phenol and/or chloroform.Researchers have long sought safer effective extraction procedures.

SUMMARY OF THE INVENTION

This invention relates to safe and effective methods for the extractionof nucleic acids. In particular, methods are described for isolatingnucleic acid from a sample containing a mixture of nucleic acids andother biological compounds, wherein the sample is combined with anextraction solution containing at least one organic compound, such asbenzyl alcohol or a benzyl alcohol derivative, forming an aqueous andnon-aqueous phase. The nucleic acid is isolated from the aqueous phase.Preferably, the resulting combined solution also contains bentonite orMacaloid, as defined below. Typically, the sample will first be combinedwith a lysing agent before extraction. The lysing agents preferred arechaotropic salts, such as guanidinium hydrochloride (GuHCl) andguanidinium isothiocyanate (GuSCN).

The extraction solution described above may also advantageously becombined with a lactam, such as cyclohexylpyrrolidone,dodecylpyrrolidone, hydroxyethylpyrrolidone, octylpyrrolidone,1-phenyl-2-pyrrolidone, and1,3-dimethyl-3,4,5,6-tetrahydro-2-(H)-pyrrolidone.

Once the nucleic acid is separated into the aqueous phase, it can beprecipitated with an alcohol, preferably ethanol or isopropanol, orconcentrated with butanol. The aqueous phase can also be used directlyin hybridization assays or immobilized on solid supports for subsequentprobing.

The organic compounds used in the present invention are of a very loworder toxicity and are not corrosive to human tissue. They retain mostof the solvent properties of phenol or phenol/chloroform, and areuniquely suited to the application of nucleic acid isolation and/orfractionation of biological macromolecular complexes. In addition, theyare substantially less expensive than the commonly employed organicsolvents. Extraction by cesium chloride, centrifugation and alcoholprecipitation may also be avoided through the use of the methods of thepresent invention, thus eliminating exposure to cesium chloride which istoxic. The extraction methods of the present invention are faster,simpler, safer and more sensitive than methods previously used in theextraction of nucleic acid. Thus, they represent a significant advancein the field, providing a safer means for scaling up the extraction ofdesired nucleic acids.

Chaotropic agents are commonly used as lysing agents in the extractionprocess. Due to the incompatibility of chaotropic agents with ionicdetergents, it has proved difficult to isolate nucleic acids fromchaotropic salt solutions. Further, it has been difficult to partitionthe nucleic acid into an aqueous phase, given the use of such high molarconcentrations of salt. The use of bentonite in combination with theextraction solutions of the subject invention in extraction techniquesinvolving chaotropic salts allows the rapid and simple recovery ofintact RNA, DNA, or total nucleic acid from particularly complexbiological samples.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the electrophoretic profile of total nucleic acids(chromosomal DNA and RNA) extracted using as the organic phase:2-methyl-benzyl alcohol in lane 1; 4-methoxybenzyl alcohol in lane 2;3-ethoxy-benzyl alcohol in lane 3; 4-phenoxy-benzyl alcohol in lane 4;benzyl-alcohol in lane 5; and phenol in lane 6.

FIG. 2 shows the electrophoretic profile of total nucleic acids fromextracted fecal samples using benzyl alcohol and bentonite. In lane 1 isshown the electrophoretic profile of total nucleic acids isolated fromfecal samples spiked with bacteria (Bacteroides gingivalis) when theextraction solution is composed of benzyl alcohol and 1% W/V bentonite.In comparison, lane 2 shows the profile when the extraction solution iscomposed of phenol. In both extractions, the recovery of total nucleicacids is comparable, and 16S and 23S rRNAs are isolated in the intactform.

DETAILED DESCRIPTION

This invention relates to novel compositions and methods for theisolation of nucleic acid from a sample containing a biological mixtureof nucleic acids and other biological compounds. The methods of thepresent invention enable one to easily process a biological samplecontaining nucleic acids by extracting nucleic acids without the use oftoxic or corrosive chemicals. The methods of the present inventionfurther enable one to prepare a nucleic acid sample for hybridizationassays.

The extraction methods include combining the sample with an extractionsolution containing at least one organic compound, as specified below,to form an aqueous and non-aqueous phase, and separating the aqueousphase from the non-aqueous phase. The extraction solution allows thesample to become biphasic. The nucleic acid is then isolated in theaqueous phase.

The extraction methods of the present invention may be applied to asample containing a biological mixture of nucleic acids (ribonucleicacid (RNA) and/or deoxyribonucleic acid (DNA)) and other biologicalcompound& which are not nucleic acids. Such a sample may include anyaqueous mixture of biological material which contains nucleic acids,including complex biological mixtures of any eucaryotic and/orprocaryotic cells, including protoplasts, or other biological materialswhich may harbor nucleic acids. The methods are thus applicable totissue culture animal cells, animal tissue (e.g., heart, liver or brain,homogenized in lysis buffer), feces, blood cells, reticulocytes,lymphocytes, plant cells or other cells sensitive to osmotic shock, andcells of bacteria, yeasts, viruses, mycoplasmas, protozoa, rickettsia,fungi and other small microbial cells and the like.

The nucleic acid sample to be assayed or to serve as a source for theextraction of nucleic acids is combined with an extraction solutionwhich allows for the separation of the nucleic acids in the sample fromthe other biological components resident in the sample. The extractionsolutions comprise benzyl alcohol, derivatives of benzyl alcohol orother solvents with similar properties. The solvent must effectivelycause the nucleic acids to be isolated from other biological componentsby partitioning the nucleic acids into an aqueous phase upon mixing theextraction solution with an aqueous sample. The extraction solution willtypically comprise an organic composition having an organic compoundwith the following properties:

(a) a dielectric constant of about 9.0 to about 15.5;

(b) a dipole moment of about 1.35 to about 1.70 coulomb-meter; and

(c) a partition coefficient of about 0.001 to about 0.3 parts water to 1part of the organic compound. Preferably such organic compound will alsohave a density of about 0.7 to about 1.9 mg/ml, preferably about 1.01 to1.9, most preferably about 1.09 to about 1.9, and will be non-corrosiveto human skin. Examples of organic compounds suitable for the extractionsolution are 4-hexylresorcinol and resorcinol, as well as those listedbelow.

The term "dielectric constant" (D) , is a dimensionless number in thecontext of Coulomb's law: ##EQU1## where F is the force of attraction orrepulsion, q1 and q2 are magnitudes of two electric charges separated bya distance d. A vacuum has a value 1.0 for D. Examples of other valuesfor D are 1.00059 for air at 1 ATM and 0° C., 24.3 for alcohol at 25° C.and 80.37 for water at 20° C.

The term "dipole moment" refers to a molecular constant (ρ or μ)indicating the distribution of electrical charges in a neutral moleculeor the magnitude of the + or - charge times the distance between thecharge centers. One coulomb-meter=2.99793×10²⁹ debye. It is zero, forexample, if they are symmetrically distributed.

The term "partition coefficient" represents the concentration of waterin the organic phase upon phase separation of equal volumes of organicsolvent and water.

The extraction solution alternatively preferably comprises at least oneorganic compound of formula I, wherein formula I is selected from thegroup of compounds having the formula C₆ H₅ CH₂ OH or: ##STR1## where:R¹ is a member selected from the group consisting of --(CH₂)_(n) OH, inwhich n is 1 to 6; --CH(OH)CH₂ OH; --CH₂ CH(OH)CH₂ OH and --CH(OH)COOR⁵,in which R⁵ is --(CH₂)_(p) CH₃, and in which p is 0 to 3;

R² is a member selected from the group consisting of --H; --F; --Cl;--Br; --I; --OH; --S(CH₂)_(r) CH₃, in which r is 0 to 3; --O(CH₂)_(q)CH₃, in which q is 0 to 6; --COOR⁶, in which R⁶ is --(CH₂)_(s) CH₃, andin which s is 0 to 3; --C₆ H₅ ; --CH₂ CH₆ H₅ ; --OC₆ H₅ ; --OCH₂ C₆ H₅ ;--CH₂ OH; --CF₃ ; and --(CH₂)_(t) CH₃, in which t is 0 to 6;

R³ is a member selected from the group consisting of --H; --F; --Cl;--Br; --I; --OH; --CH₃ ; --(CH₂)_(x) CH₃, in which x is 0 to 3;--O(CH₂)_(v) CH₃, in which v is 0 to 3; and

R⁴ is a member selected from the group consisting of --H; --F; --Cl;--Br; --I; --(CH₂)_(w) CH₃, in which y is 0 to 3; --O(CH₂)_(w) CH₃, inwhich w is 0 to 3; and further,

wherein R², R³ and R⁴ may occur in any position in relation to R¹,provided that:

(a) when R¹ is --CH(OH)CH₂ OH, --CH₂ CH(OH)CH₂ OH or --CH(OH)COOR⁵, then

R², R³ and R⁴ must be --H;

(b) when R¹ is --CH₂ OH and R² is --C₆ H₅, --OCH₂ C₆ H₅, or --CH₂ C₆ H₅,R³ must be --CH₃, --OH, --Cl, --Br, --F, --I, --H, --OCH₃ or --OCH₂ CH₃,and R⁴ must be --H; and

(c) when R¹ is --(CH₂)_(n) OH and n is 2 to 6, then R³ and R⁴ must bothbe --H.

Formula I is intended to include all isomer forms of the alkyls, bothbranched and unbranched. Unless otherwise stated, all number ranges areinclusive of the stated range, with the use of zero referring to theabsence of the element. For example, --(CH₂)_(n) CH₃, in which n is 0 to2, refers to the three components: --CH₃, --CH₂ CH₃ and --CH₂ CH₂ CH₃.HC₆ H₅ is a benzene ring.

The following compounds of formula I are particularly preferred:

those where: R¹ is --(CH₂)_(n) OH, in which n is 1 to 4, and R², R³ andR⁴ are all --H;

where: R¹ is --(CH₂)_(n) OH, in which n is 1 to 4;

R² is --(CH₂)_(t) CH₃, in which t is 0 to 4; and

R³ and R⁴ are both --H; or

where: R¹ is --(CH₂)_(n) OH, in which n is 1 to 4;

R² is --O(CH₂)_(q) CH₃, in which q is 0 to 4; and

R³ and R⁴ are both --H.

The above compounds are all commercially available. Representativecompounds may be purchased from Aldrich Chemical Company, Inc.,Milwaukee, Wisc., or Fluka Chemical Company, New York, N.Y.

The organic compounds or mixtures thereof designated above are thosewhich have a density of about 0.7-1.9 g/ml, preferably greater thanabout 1.01, most preferably greater than about 1.09. They will also havea melting point of 37° C. or less.

The most preferred organic compounds of the extraction solution arebenzyl alcohol, 2-methyl-benzyl alcohol, 4-methoxy-benzyl alcohol,3-ethoxy-benzyl alcohol and 4-phenoxy-benzyl alcohol.

Some of the above organic compounds are not in liquid form, and may bedissolved in an appropriate solvent such as benzyl alcohol for use inthe extraction solution.

Conventionally, organic solvents, such as phenol or a phenol-chloroformcombination, are used to extract nucleic acid, using a phase separation.These methods may be used effectively with the extraction solutions ofthe present invention; however, an advantage of the methods of thepresent invention is that such toxic compounds and tedious extractionmethods are not necessary.

The extraction solution is combined with the sample containing abiological mixture so that the combined solution becomes biphasic, withthe non-nucleic acid material present in the organic phase and thenucleic acid material present in the aqueous phase. It is useful tosaturate the organic phase with an appropriate buffer, typically thebuffer used to dilute or suspend the sample. The combined solution istypically mixed and subjected to low speed centrifugation one or moretimes. Centrifugation is not necessary for the phase separation, butprovides for a faster separation.

The nucleic acid in the aqueous phase is precipitated out with analcohol such as ethanol or isopropanol, in the manner known to thoseskilled in the art. It may also be left in the aqueous phase, forexample, for use in hybridization procedures. The aqueous phase may alsobe reextracted by adding fresh extraction solution.

The extraction solution will be combined with the sample such that thecombined solution will typically contain about 10 to about 80%,preferably about 40 to about 60% and most preferably about 50%(volume:volume basis) of the organic composition as described above.Preferably then, the extraction solution will also contain standardbuffers and detergents to promote lysing of cells. A buffer such assodium citrate, Tris--HCl, PIPES or HEPES, preferably Tris--HCl at aconcentration of about 0.01 to 0.1 M, can be used. The buffer willtypically also contain about 0.05 to 5% of an ionic or nonionicdetergent, such as sodium dodecylsulfate (SDS) or sarkosyl (SigmaChemical Co., St. Louis, Mo.), between 1 to 20 mM EDTA, and betweenabout 0-250 mM salt, such as NaCl.

Also preferably resident in the extraction solution is a nucleaseinhibitor, preferably an organoclay or the like, and more preferablybentonite, Macaloid®, Bentone® (a bentonite or hectorite organoclayplatelet having a long chain organic compound bonded to its two faces)or the like, as well as combinations, derivatives or analogs thereof, ata concentration of about 0.1 to about 10%, preferably about 1 to about5% (on a weight:volume basis). Bentonite is intended to encompass hereany clay or silicate, such as diatomaceous earth, or any substanceconsisting primarily of montmorillonite (Al₂ O₃ ·4SiO₂ ·H₂ O), andtypically aluminum silicate or the like. To use, the bentonite is firstwater-saturated and added to the extraction solution. Macaloid® (a clay)and Bentone® are available from N.L. Chemicals, Hightstown, N.J.Bentone® is particularly suitable for use within the present inventionbecause the particulate matter remains in relatively homogeneoussuspension for extended periods of time, as compared to bentonite andMacaloid®. It may be necessary to first purify the nuclease inhibitor,for instance bentonite or Macaloid®, as described in Sambrook et al.,Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1989), which is incorporated by referenceherein. For efficient extraction, it is preferred that small uniformparticles be used. Such nuclease inhibitors are particularly desirablewhen extraction of RNA is desired. In samples where ribonuclease doesnot substantially hinder extraction, or if DNA only is being extracted,nucleic acid may be extracted without the use of such nucleaseinhibitors.

The extraction solution optionally includes an aminotriarylmethane dye,such as those described in R. D. Lillie, H. J. Conn's Biological Stains,Williams & Wilkins, Baltimore, Md. (1977). Properties of suitable dyesinclude solubility in organic solvents, minimal or no partitioning intowater, and minimal or no binding to nucleic acids. A preferred dye ismethyl violet, with methyl violet 6B particularly preferred. Theinclusion of dye in the extraction solution results in a colored organicphase and a colorless aqueous phase, thereby provided enhanceddelineation of the two phases during the extraction process. The dyeconcentration is sufficient to provide visual contrast between theaqueous and organic phases, and typically is in the range of about0.0005-0.01% (w/v). Because the dye is resident in the organic phase,the preferred organic compound may vary with choice of dye.

The biological samples are typically lysed through the use of lysingagents, prior to or during extraction, to disturb the protein structureand to release the nucleic acid. Preferably, the sample will besubjected to lysis prior to the addition of the extraction solution. Anylysing agents typically used in nucleic acid extraction procedures areappropriate, such as SDS, lysozyme, Proteinase K in various combinationswith chaotropic agents, or any other agent which would weaken or disruptthe integrity of the cell membrane.

Chaotropic agents, which disturb the secondary and tertiary structure ofproteins (for example, guanidinium salts such as guanidiniumhydrochloride (GuHCl), guanidinium isothiocyanate (GuSCN), urea or otherisothiocyanates), may be used as lysing agents, in combination with orprior to the extraction solution, to dissociate nucleic acids andinhibit nucleases. The use of chaotropic agents in the extraction andhybridization of nucleic acids is described in E.P Publication No. 0 127327, which is incorporated by reference herein. The chaotropic agent ispresent at a concentration sufficient to release nucleic acid fromtarget cells and to protect the released nucleic acid from nucleases.Typically, the chaotrope is present at a concentration from about 1 M toabout 5 M, and more preferably is present at about 2 M to about 3 M.Lactams may also be used in combination with the extraction solution tofacilitate lysing and extraction. Examples of lactams and their use inextraction procedures are thoroughly described in commonly assigned U.S.Ser. No. 07/384,235, filed on Jul. 24, 1989, now abandoned which isincorporated by reference herein. Preferred lactams arecyclohexylpyrrolidone, 1 -phenyl-2-pyrrolidone and1,3-dimethyl-3,4,5,6-tetrahydro-2-(H)-pyrrolidone.

For the extraction of total nucleic acid (DNA and RNA), extractiontypically takes place at about 37° C. to about 65° C. for about 1 to 10minutes. The extraction procedure will preferably yield an aqueoussample having a concentration of nucleic acid such that after combiningthe sample with the extraction solution, the amount of nucleic acidpresent in the aqueous phase will not be in excess of 1 mg/ml. A usefulrule of thumb is to not permit the total biological material to exceed50 mg/ml in a given sample. The ratio of sample volume to extractionsolution volume is typically 0.5:1 to 3:1, more typically 0.75:1 to 2:1,most typically about 1:1.

Extraction of Ribosomal RNA

Extraction solutions of the present invention also permit the selectiveextraction of ribosomal RNA (rRNA). After the sample containing thenucleic acids has been lysed, the sample is heated prior to or aftercombining the sample with the extraction solution. The yield of rRNA isincreased 5 to 50 fold by heating the sample at about 65° C. for about10 minutes before or after addition of the extraction solution. If noheat is used, DNA is primarily extracted.

Thus, a sequential extraction of rRNA and DNA may be accomplished byfirst lysing the sample. Then, extraction is performed at roomtemperature using the extraction solutions as described herein, and DNAis isolated from the first aqueous phase, the rRNA remaining in thefirst organic phase. The rRNA is then extracted from the first organicphase after the addition of a standard buffer solution (e.g., 1% SDS, 50mM Tris, 25 mM EDTA and 0.05 mM NaCl). This solution is heated,typically to about 65° C., to create a second organic phase and secondaqueous phase.

Extraction solutions which are most preferred and which may bealternatively offered in kit form are those extraction compositionswhich comprise bentonite or Bentone®, a lysing agent, and an organiccompound selected from those described above in connection with theextraction solutions. The novel extraction compositions may include incombination any of the elements described above, which may be includedin the variously described extraction solutions.

Extraction to Improve Hybridization

It is also advantageous to use the extraction methods and compositionsof this invention prior to conducting a hybridization assay on a complexbiological sample, such as on feces or blood. The extraction proceduremay be necessary in some cases to remove contaminants which contributeto background interference. Extraction followed by procedures toconcentrate the nucleic acid may improve sensitivity and thesignal-to-noise ratio in hybridization assays.

A hybridization assay according to the present invention can beperformed by any method known to those skilled in the art or that isanalogous to immunoassay methodology given the guidelines presentedherein. Preferred methods of assay are the sandwich assays andvariations thereof, and the competition or displacement assay.Hybridization techniques are generally described in "Nucleic AcidHybridization, A Practical Approach," Ed Hames, B. D. and Higgins, S.J., IRL Press, 1985; Gall and Pardue (1969), Proc. Natl. Acad. Sci.U.S.A. 63:378-383; and John, Burnsteil and Jones (1969) Nature223:582-587. As improvements are made in hybridization techniques, theycan readily be applied.

Sandwich assays are commercially useful hybridization assays fordetecting or isolating nucleic acid sequences. Such assays utilize a"capture" nucleic acid covalently immobilized to a solid support andlabelled "signal" nucleic acid in solution. The clinical sample willprovide the target nucleic acid. The "capture" nucleic acid and "signal"nucleic acid probe hybridize with the target nucleic acid to form a"sandwich" hybridization complex. To be effective, the signal nucleicacid cannot hybridize with the capture nucleic acid.

In a hybridization assay, the target nucleic acid is the nucleotidesequence of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) orribosomal ribonucleic acid (rRNA), whose presence is of interest andwhose presence or absence is to be detected. The target nucleic acid maybe provided in a complex biological mixture of nucleic acid (RNA, DNAand/or rRNA) and non-nucleic acid.

The hybridization media may be pre-prepared, either commercially or inthe laboratory, to contain all the necessary components forhybridization. For instance, in a sandwich assay the media couldcomprise a lactam, desired buffers and detergents, a capture nucleicacid bound to a solid support such as a microbead, and a signal nucleicacid. This media then need only be combined with the solution containingthe target nucleic acid at the time the assay is to be performed. Oncehybridization occurs, the hybridization complex attached to the solidsupport may be washed and the extent of hybridization determined.

Once the appropriate sequences are determined, DNA probes are preferablychemically synthesized using commercially available methods andequipment. For example, the solid phase phosphoramidite method can beused to produce short probes of between 15 and 50 bases and having amolecular weight of less than 16,000 daltons. (Caruthers et al., ColdSpring Harbor Symp. Quant. Biol. 47:411-418 (1982), and Adams et al., J.Am. Chem. Soc. 105:661 (1983)). When synthesizing a probe for a specifictarget, the choice of nucleotide sequence will determine the specificityof the test. For example, by comparing DNA sequences from several virusisolates, one can select a sequence for virus detection that is eithertype specific or genus specific. Comparisons of DNA regions andsequences can be achieved using commercially available computerprograms.

The determination of the extent of hybridization may be done by any ofthe methods well-known in the art. If there is no detectablehybridization, the extent of hybridization is thus 0. Typically,labelled signal nucleic acids are used to detect hybridization.Complementary nucleic acids or signal nucleic acids may be labelled byany one of several methods typically used to detect the presence ofhybridized polynucleotides. The most common method of detection is theuse of autoradiography with ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C, or ³² P labelledprobes or the like. The choice of radioactive isotope depends onresearch preferences due to ease of synthesis, varying stability, andhalf lives of the selected isotopes. Other labels include ligands whichbind to labelled antibodies, fluorophores, chemiluminescent agents,enzymes, and antibodies which can serve as specific binding pair membersfor a labelled ligand. The choice of label depends on sensitivityrequired, ease of conjugation with the probe, stability requirements,and available instrumentation.

Kits for the extraction of and hybridization of nucleic acids are alsocontemplated, which comprise the above solutions and compositions invarious desired combinations.

Typically, compounds present in biological samples exert an inhibitoryeffect on target amplification procedures, such as PCR and LCR. Thisobserved inhibition is particularly problematic with amplification ofnucleic acid extracted from blood samples. The methods of the presentinvention remove most, if not all, inhibitors of target amplificationfrom biological samples in general, and particularly from blood. Thus,the claimed methods may be advantageously used in combination withtarget amplification procedures. The present invention is particularlysuited to target amplification of nucleic acid obtained from biologicalsamples containing low copy number target nucleic acid.

The following examples are offered by way of illustration and are not tobe construed as limiting the invention, as claimed, in any way.

EXAMPLES Example 1 Extraction of total nucleic acids with benzyl alcoholand benzyl alcohol derivatives

This extraction protocol allows the isolation of nucleic acids fromsamples lysed with guanidinium isothiocyanate without the use of phenolor phenol/chloroform. The organic phase is composed of benzyl alcohol ora derivative of benzyl alcohol.

Approximately 5×10⁹ Bacteroides gingivalis cells were lysed in 750 μl of3 M GuSCN (Kodak, Rochester, N.Y.) lysing solution-containing 2%sarkosyl (Sigma Chemical Company, St. Louis, Mo.), 50 mM Tris (pH 7.6),and 25 mM EDTA. Then 100 μl of the lysate was aliquoted equally into6-1.5 ml microcentrifuge tubes. To each tube was added respectively: 500μl of organic phase saturated with 0.05 M Tris--HCl, 5 mM EDTA (pH 7.2),where the organic phase in:

tube 1 was 2-methyl-benzyl alcohol (Aldrich Chemical Company, Milwaukee,Wisc.);

tube 2 was 4-methoxy-benzyl alcohol (Aldrich);

tube 3 was 3-ethoxy-benzyl alcohol (Aldrich);

tube 4 was 4-phenoxy-benzyl alcohol (Aldrich);

tube 5 was benzyl alcohol (Aldrich); and

tube 6 was phenol (Bethesda Research Laboratories (BRL), Gaithersburg,Md.);

and 250 μl of extraction buffer (0.05 M NaCl, 50 mM Tris--HCl (pH 7.2),5 mM EDTA and 0.5% sodium dodecyl sulfate (Sigma Chemical Company, St.Louis, Mo. (SDS)). The solutions were vigorously mixed for 15 secondsand heated at 65° C. for 10 minutes. The tubes were then againvigorously mixed for 15 seconds. The phases were then separated bycentrifugation at 10,000 rpm in a microcentrifuge. The upper aqueousphase was removed (generally a volume of 400 μl ) and then twice thevolume of 100% ethanol was added to the aqueous phase. Precipitation ofthe nucleic acid was allowed to occur for 5 minutes at 19° C. Thenucleic acids were then pelleted from solution by centrifugation at10,000 rpm for 10 minutes. The liquid phase was then decanted anddiscarded. The nucleic acid pellet was dissolved in 100 μl of distilledwater and then subjected to agarose gel electrophoresis. The results arepresented in FIG. 1, and indicate that the extractions were allcomparable in yield and purity to those using phenol.

Example 2 Extraction of total nucleic acids from fecal samples usingbenzyl alcohol and bentonite

This extraction protocol allows the isolation of nucleic acids fromparticularly complex samples lysed with guanidinium isothiocyanatewithout the use of phenol or phenol/chloroform. The organic phase iscomposed of benzyl alcohol and bentonite.

Fecal samples in transport medium (Trend Fekal® Enteric Plus TransportSystem, Trend Scientific Inc., St. Paul, Minn.) were aliquoted into 100μl portions. Each aliquot was lysed with 300 μl of GuSCN lysis solution,as in Example 1, and were spiked with 5×10⁸ Bacteriodes gingivaliscells. Following spiking, the samples were divided into two equal sets.One set was extracted using phenol, and the other set was extracted byadding 500 μl of a benzyl alcohol/bentonite solution which comprised:benzyl alcohol, containing 1% W/V H₂ O-saturated bentonite (Sigma) and250 μl of extraction buffer (0.05 M NaCl, 50 mM Tris--HCl (pH 7.2), 5 mMEDTA and 0.5% sodium dodecyl sulfate). The solutions were, vigorouslymixed for 15 seconds and heated at 65° C. for 10 minutes. The tubes werethen again vigorously mixed for 15 seconds. The phases were thenseparated by centrifugation at 10,000 rpm for 2 minutes in amicrocentrifuge. The upper aqueous phase was removed (generally a volumeof 400 μl ) and then twice the volume of 100% ethanol was added to theaqueous phase. Precipitation of the nucleic acid was allowed to occurfor 5 minutes at 19° C. The nucleic acids were then pelleted fromsolution by centrifugation at 10,000 rpm for 10 minutes. The liquidphase was then decanted and discarded. The nucleic acid pellet wasdissolved in 100 μl of distilled water and then subjected to agarose gelelectrophoresis.

For the phenol extraction, samples were lysed as above and extractedexactly as described for the benzyl alcohol procedure. The phases wereseparated by centrifugation as above, and the aqueous phase re-extractedat 65° C. with an additional volume of phenol. The isolated nucleicacids were then precipitated with 70% ethanol, as above. The liquidphase was decanted and discarded. The nucleic acid pellet was dissolvedin 100 μl of distilled water and then subjected to agarose gelelectrophoresis. The results are presented in FIG. 2, which shows thattotal nucleic acids may be isolated from sources with high nucleaselevels using the benzyl alcohol extraction.

Example 3 Improved hybridization procedures by pre-extraction of acomplex biological sample

The assay utilizes nylon solid supports in a sandwich assay format inwhich a target nucleic acid sequence is sequestered and then detectedusing a fluorescence-based assay format.

Fecal samples in transport medium (Trend Fekal™ Enteric Plus TransportSystem, Trend Scientific Inc.) were aliquoted into 100 μl portions. Eachaliquot was lysed with 300 μl of GuSCN lysis solution as in Example 1,and was spiked with 5×10⁸ Bacteroides gingivalis. Following spiking, thesamples were divided into two equal aliquots. One aliquot was extractedusing the benzyl alcohol/bentonite method, as described above in Example2, and the other aliquot was not subjected to extraction. The nucleicacid pellet from the extracted sample was dissolved into 100 μl of 3MGuSCN lysis solution (in 2% sarkosyl, 50mM Tris (pH 7.6) and 25mM EDTA).Both the unextracted lysate and the isolated nucleic acid in 3M GuSCNwere heated to 65° C. for 5 minutes. A biotinylated 24-meroligonucleotide probe complementary to conserved regions of bacterial16S rRNA (signal probe) was added to a final concentration of 100nanograms per ml to the lysate and to the isolated nucleic acid.

5-fold serial dilutions of the lysates were made using diluents in the3M GuSCN lysing solution containing the biotinylated signaloligonucleotides:

Oligonucleotide sequences:

Bg1: 5'-XCAATACTCGTATCGCCCGTTATTC-3'

UP9A: 5'-XCTGCTGCCTCCCGTAGGAGT-3'

and 1×10⁸ total cells of Actinobacillus actinomycetecomitans,Bacteroides intermedius, Eikenella corrodens, Wolinella recta, andFusobacterium nucleatum in the case of the extracted sample and thefecal lysate in the case of the unextracted sample. The diluentscontained 100 mg/ml biotinylated signal oligonucleotide, as describedabove. The solutions were then incubated for 30 minutes at ambienttemperature with 2 nylon beads prepared by The Hoover Group (Sault St.Marie, Mich.) that had covalently immobilized 0.1 μg of Bg1-specificoligonucleotide probe (capture probe). The solid supports were washedwith SDS/FW (0.09M NaCl, 50 mM Tris pH 7.6, 25mM EDTA and 0.1% SDS) atambient temperature, following by washing with 0.5% Tween 20®(Pierce,Rockford, Ill.), 1 mM MgCl₂, 0.01 M Tris--HCl pH 8.0 (APB), and thenincubated with 0.4 μg/ml of streptavidin/alkaline phosphatase (SA/AP)conjugate in APB for 5 minutes at ambient temperature. The solidsupports were then washed 5 times with APB, TMNZ (0.05 M Tris (pH 9.5),1 mM MgCl₂, 0.5 mM ZnCl₂), and then the presence of alkaline phosphatasewas determined by incubating the nylon beads with 150 μl of 0.5 mM4-methyl-umbelliferyl phosphate (4-hydroxy-methyl coumarin) in blackmicrotiter well strips (Dynatek Laboratories, Chantilly, Va.).Incubation was for 30 minutes at 37° C. The solution was then decantedand placed in a 96 well microtiter plate. The plates were then directlyread using a Fluoroskan II fluorometer (Flow Laboratories, McLean, Va.)using an excitation wavelength of 360 nm and an emission wavelength of456 nm. The results are shown in Table 1 below.

                  TABLE 1                                                         ______________________________________                                        Fluorescent Signal                                                            Cell number:                                                                            Extracted sample                                                                            Non-Extracted Sample                                  ______________________________________                                          1 × 10.sup.8                                                                    1876          2100                                                    2 × 10.sup.7                                                                    1350          2100                                                    4 × 10.sup.6                                                                    740             1550**                                                8 × 10.sup.5                                                                    220           1180                                                  1.6 × 10.sup.5                                                                    56             980                                                  3.2 × 10.sup.4                                                                    29            1210                                                  6.4 × 10.sup.3                                                                      23**        1160                                                  control   18             940                                                  ______________________________________                                         where ** indicates the lowest level of detection.                        

The results indicate that, in the 30 minute hybridization, a level of6×10³ cells was detected using the extracted sample, whereas with theunextracted sample a level of only 4×10⁶ cells were detected.

Example 4 Extraction of DNA from biological samples using benzyl alcoholand Bentone®

A suspension of E. coli was harvested by centrifugation, and the pelletwas resuspended to between 10⁸ and 10¹⁰ cells/ml in 50 mM Tris buffer(pH 7.6) containing 10 mM EDTA and 10% (w/v) sucrose. The suspension wasthen incubated for 5-15 minutes at ambient temperature. Alternatively, ablood sample was collected in a collection tube containing sodium EDTA.

The biological sample (either the bacterial suspension or the bloodsample) was lysed with an equal volume of lysis buffer (5 M GuSCN, 83 mMTris--HCl, pH 7.6, 17 mM EDTA and 3.3% (w/v) sarkosyl).

The lysate (200 μl ) was transferred to a 2 ml microcentrifuge tube.After vigorous shaking, 700 μl of an extraction solution (1.1% (w/v)Bentone® in 99% pure benzyl alcohol) was added to the lysate. To thismixture was added 400 μl buffer (50 mMTris--HCl, pH 7.6, 10 mM EDTA, 100mM NaCl, 0.5% (w/v) SDS). The mixture was vortexed 10 seconds, thencentrifuged at 12,000×g for 5 minutes. The upper aqueous phase wastransferred to a new 2 ml microcentrifuge tube and 0.1 volume 3 M sodiumacetate was added. An equal volume of isopropanol was added, and thesolution mixed gently to precipitate DNA. After centrifugation at12,000×g for 10 minutes, the supernatant was discarded and 1 ml of 70%ethanol was added to the pelleted DNA. After gentle mixing, thepreparation was centrifuged at 12,000×g for 5 minutes. The supernatantwas discarded, the pellet was air dried, and the DNA pellet was thenresuspended in RNase-free water or appropriate buffer to a desiredconcentration.

DNA extracted according to the protocol set forth in this Example issuitable for use in target amplification procedures, such as PCR(polymerase chain reaction). Various target amplification procedures arewell known in the art. Table 2 below shows that the DNA extractionprocedure of this Example permits amplification and detection of lowcopy number target nucleic acid. Briefly, known concentrations-of anHIV-1 plasmid were added to human whole blood to give 0-250 pHIV-1copies per PCR reaction. Duplicate 100 μl blood samples were extracted,the extracted nucleic acid was resuspended in 100 μl of water, and 50 μlof this nucleic acid suspension were used per amplification reaction.Amplified target was detected by hybridization of a complementary ³²P-labeled oligonucleotide probe.

                  TABLE 2                                                         ______________________________________                                        Amplification and Detection                                                   of Low Copy Number Target DNA                                                 pHIV-1 Copies per  Signal                                                     PCR Reaction       Detected                                                   ______________________________________                                        250                +                                                          100                +                                                          50                 +                                                          20                 +                                                          10                 +                                                           5                 +                                                           2                 -                                                           0                 -                                                          Negative PCR controls                                                                            -                                                          ______________________________________                                    

Table 3 below compares extraction of DNA from whole blood using aProteinase K/phenol extraction method versus the extraction method ofthis Example. Briefly, three PCR-positive HTLV-I blood samples and fivecontrol blood samples (HTLV-I negative by PCR) were collected in tubescontaining sodium EDTA. Each of the positive samples was seriallydiluted in 5-fold steps with the control blood. Duplicate 100 μl bloodsamples were extracted using a Proteinase K/phenol procedure (see, forexample, Sambrook et al., in "Molecular Cloning. A Laboratory Manual",Cold Spring Harbor Laboratory Press, N.Y., 1989; R. Higuchi, in"Amplifications. A Forum for PCR Users", Perkin-Elmer Corp , Norwalk,Conn., 1989) or the method of this Example. The extracted nucleic acidwas resuspended in 100 μl of water, and 50 μl of this nucleic acidsuspension were used per PCR reaction. Amplified target was detected byhybridization of a complementary ³² P-labeled oligonucleotide probe.Table 3 demonstrates that Proteinase K/phenol and the describedprocedure provide comparable limits of extraction and detection.

                  TABLE 3                                                         ______________________________________                                        Comparison of Proteinase K/Phenol                                             and the Extraction Procedure of Example 4 -                                   PCR Amplification and Detection                                               of Nucleic Acid From Whole Blood                                              Dilution Factor of                                                                             Signal Detected                                              HTLV-I Positive  Proteinase K/                                                Blood Sample     Phenol     Example 4                                         ______________________________________                                         1               +          +                                                  5               +          +                                                  25              +          +                                                 125              +          +                                                 625              -          -                                                 3125             -          -                                                 HTLV-I Negative Blood                                                                          -          -                                                 Negative PCR Controls                                                                          -          -                                                 ______________________________________                                    

Example 5 Extraction of total nucleic acid from biological samples usingbenzyl alcohol and Bentone®

A suspension of E. coli was harvested by centrifugation, and the pelletwas resuspended to between 10⁸ and 10¹⁰ cells/ml in 50 mM Tris buffer(pH 7.6) containing 10 mM EDTA and 10% (w/v) sucrose. The suspension wasthen incubated for 5-15 minutes at ambient temperature. Alternatively, ablood sample was collected in a collection tube containing sodium EDTA.

The biological sample (either the bacterial suspension or the bloodsample) was lysed with an equal volume of lysis buffer (5 M GuSCN, 83mMTris--HCl, pH 7.6, 17 mM EDTA and 3.3% (w/v) sarkosyl).

The lysate (200 μl) was transferred to a 2 ml microcentrifuge tube.After vigorous shaking, 700 μl of an extraction solution (1.1% (w/v)Bentone® in 99% pure benzyl alcohol) was added to the lysate. To thismixture was added 400 μl buffer (50 mM Tris--HCl, pH 7.6, 10 mM EDTA,100 mM NaCl, 0.5% (w/v) SDS). The mixture was vortexed 10 seconds,heated for 10 minutes at 65° C. with occasional mixing, and thencentrifuged at 12,000×g for 5 minutes. The upper aqueous phase wastransferred to a new 2 ml microcentrifuge tube, and 500 μl extractionsolution (benzyl alcohol and Bentone®, as described above) were added.The mixture was vortexed 10 seconds and centrifuged at 12,000×g for 5minutes. The upper aqueous phase was again transferred to a new 2 mlmicrocentrifuge tube, and 0.1 volume 3 M sodium acetate was added. Anequal volume of isopropanol was added, and the solution mixed gently toprecipitate total nucleic acid. After centrifugation at 12,000×g for 10minutes, the supernatant was discarded and 1 ml of 70% ethanol was addedto the pelleted nucleic acid. After gentle mixing, the preparation wascentrifuged at 12,000×g for 5 minutes. The supernatant was discarded,the pellet was air dried, and the nucleic acid pellet was thenresuspended in RNase-free water or appropriate buffer to a desiredconcentration.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       NCAATACT CGTATCGCCCGTTATTC25                                                  (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       NCTGCT GCCTCCCGTAGGAGT21                                                  

What is claimed is:
 1. An extraction composition for the isolation ofreleased nucleic acids comprising:at least one organic compound which isa liquid at room temperature and which is selected from the group ofcompounds having the formula C₆ H₅ CH₂ OH or: ##STR2## where: R¹ is--CH₂ OH, R² is a member selected from the group consisting of --H, --F,--C, --Br, --I, --OH, --S(CH₂)_(r) CH₃ in which r is 0 to 3,--O(CH₂)_(q) CH₃ in which q is 0 to 6, --COOR⁶ in which R⁶ is--(CH₂)_(s) CH₃ in which s is 0 to 3, --C₆ H₅, --CH₂ C₆ H₅ , --OC₆ H₅,--OCH₂ C₆ H₅, --CH₂ OH, --CF₃, and --(CH₂)_(t) CH₃ in which t is 0 to 6and R³ and R⁴ are both --H; or R¹ is --CH₂ OH, R² and R³ are the samemember selected from the group consisting of --H, --F, --Cl, --Br, --I,--OH, --(CH₂)_(x) CH₃ in which x is 0 to 3, --O(CH₂)_(v) CH₃ in which vis 0 to 3 and R⁴ is --H; or R¹ is --CH₂ OH, R², R³ and R⁴ are the samemember selected from the group consisting of --H, --F, --Cl, --Br, --I,--(CH₂)_(y) CH₃ in which y is 0 to 3, --O(--CH₂)_(w) CH₃ in which w is 0to 3; or R¹ is --CH₂ OH, R² is --C₆ H₅, --OCH₂ C₆ H₅, or --CH₂ C₆ H₅, R³is --CH₃, --OH, --Cl, --Br, --F, --I, --H, --OCH₃ or --OCH₂ CH₃ and R⁴is --H; and a clay or a silicate or an admixture of said clay and saidsilicate; wherein said clay or said silicate or said admixture is 0.1%to about 5% by weight of said composition.
 2. The composition of claim 1wherein R² is --C₆ H₅, --OCH₂ C₆ H₅, or --CH₂ C₆ H₅,R³ must be --CH₃,--OH, --Cl, --Br, --F, --I, --H, --OCH₃ or --OCH₂ CH₃ and R⁴ must be--H.
 3. The composition of claim 1 wherein the clay or silicate ismontmorillonite clay, an organic derivative of montmorillonite clay,diatomaceous earth, aluminum silicate or a combination thereof.
 4. Thecomposition of claim 1 wherein the organic compound is selected from thegroup consisting of benzyl alcohol, 3-ethoxy-benzyl alcohol and4-phenoxy-benzyl alcohol.
 5. The composition .of claim 1 wherein theorganic compound or combination thereof is one which has a density of1.01 g/ml or greater.
 6. The composition of claim 1 further including adye.
 7. The composition of claim 1 wherein the organic compound issaturated with an aqueous solution.
 8. The composition of claim 7wherein the aqueous solution is a buffer solution.
 9. The composition ofclaim 7 wherein the aqueous solution includes detergent at aconcentration of about 0.05% to about 5%.
 10. The composition of claim 7wherein the aqueous solution includes EDTA at a concentration of about 1to about 20 mM.
 11. The composition of claim 7 wherein the aqueoussolution includes a salt at a concentration of about 1 to about 250 mM.12. The composition of claim 7 wherein the aqueous solution includes achaotropic agent at a concentration of about 1 to about 5 M.
 13. Anextraction composition for the isolation of released nucleic acidscomprising:a clay or a silicate or an admixture of said clay and saidsilicate; and at least one organic compound which is selected from thegroup of compounds having the formula C₆ H₅ CH₂ OH or: ##STR3## where:R¹ is --CH₂ OH; R² is a member selected from the group consisting of--H, --F, --Cl, --Br, --I, --OH, --S(CH₂)_(r) CH₃ in which r is 0 to 3,--O(CH₂)_(q) CH₃ in which q is 0 to 6, --COOR⁶ in which R⁶ is--(CH₂)_(s) CH₃ in which s is 0 to 3, --C₆ H₅, --CH₂ C₆ H₅, --OC₆ H₅,--OCH₂ C₆ H₅, --CH₂ OH, --CF₃, and --(CH₂)_(t) CH₃ in which t is 0 to 6and R³ and R⁴ are both --H; or R¹ is --CH₂ OH, R² and R³ are the samemember selected from the group consisting of --H, --F, --Cl, --Br, --I,--OH, --(CH₂)_(x) CH₃ in which x is 0 to 3, --O(CH₂)_(v) CH₃ in which vis 0 to 3 and R⁴ is --H; or R¹ is --CH₂ OH, R², R³ and R⁴ are the samemember selected from the group consisting of --H, --F, --Cl, --Br, --I,--(CH₂)_(y) CH₃ in which y is 0 to 3, --O(CH₂)_(w) CH₃ in which w is 0to 3; or R¹ is --CH₂ OH, R² is --C₆ H₅, --OCH₂ C₆ H₅, or --CH₂ C₆ H₅, R³is --CH₃, --OH, --Cl, --Br, --F, --I, --H, --OCH₃ or --OCH₂ CH₃ and R⁴is --H; and wherein the organic compound selected is one that is a solidat room temperature which is dissolved in an organic solvent.
 14. Thecomposition of claim 13 wherein when R² is --C₆ H₅, --OCH₂ C₆ H₅, or--CH₂ C₆ H₅,R³ must be --CH₃, --OH, --Cl, --Br, --F, --I, --H, --OCH₃ or--OCH₂ CH₃ and R⁴ must be --H.
 15. The composition of claim 13 whereinthe clay or silicate is montmorillonite clay, an organic derivative ofmontmorillonite clay, diatomaceous earth, aluminum silicate or acombination thereof.
 16. The composition of claim 13 wherein the clay orsilicate or the admixture of the clay and the silicate is present at aconcentration of about 0.1% to about 5%.
 17. The composition of claim 13wherein the organic compound is 4-methoxy-benzyl alcohol.
 18. Thecomposition of claim 13 wherein the organic compound or combinationthereof is one which has a density of 1.01 g/ml or greater.
 19. Thecomposition of claim 13 further including a dye.
 20. The composition ofclaim 13 wherein the organic compound is saturated with an aqueoussolution.
 21. The composition of claim 20 wherein the aqueous solutionis a buffer solution.
 22. The composition of claim 20 wherein theaqueous solution includes detergent at a concentration of about 0.05% toabout 5%.
 23. The composition of claim 20 wherein the aqueous solutionincludes EDTA at a concentration of about 1 to about 20 mM.
 24. Thecomposition of claim 20 wherein the aqueous solution includes salt at aconcentration of about 1 to about 250 mM.
 25. The composition of claim20 wherein the aqueous solution includes a chaotropic agent at aconcentration of about 1 to about 5 M.